Expression and Characterization of the TNT Nitroreductase of Pseudomonas sp. HK-6 in Escherichia coli |
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Authors: | Bheong-Uk Lee Sung-Chul Park Yun-Seok Cho Hyung-Yeel Kahng Kye-Heon Oh |
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Affiliation: | (1) Division of Biological Sciences, Kosin University, Busan, 606-701, Republic of Korea;(2) Department of Biotechnology, Soonchunhyang University, Asan, Chung-Nam, 336-745, Republic of Korea;(3) Department of Environmental Education, Sunchon National University, Sunchon, Jeon-Nam, 540-742, Republic of Korea |
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Abstract: | Pseudomonas sp. HK-6 is able to utilize 2,4,6-trinitrotoluene (TNT) as a sole nitrogen source. The pnrB gene of the HK-6 strain was cloned using degenerate primers synthesized on the basis of the sequence information of the terminal amino acids of a previously purified native TNT nitroreductase. The nucleotide sequence of pnrB was 654 bp long, and its deduced polypeptide sequence was composed of 217 amino acid residues with a predicted molecular mass of 24 kDa. To facilitate the purification and characterization of this enzyme, an Escherichia expression plasmid harboring six histidine residues fused to a pnrB gene was constructed (His6-PnrB) and designated pPSC1. The His6-PnrB induced in E. coli BL21 was purified using a nickel affinity column to homogeneity. Its enzymatic activity was assayed by measuring absorbance changes at 340 nm due to NADH oxidation. The V max and K m values of the enzyme for TNT were 12.6 μmol/min/mg protein and 2.9 mM, respectively. In addition, the pnrB knockout mutant was constructed via a single-crossover homologous recombination with a partial pnrB DNA fragment that lacked both start and stop codons. Eight days was required for complete degradation of 0.5 mM TNT by the wild-type HK-6 strain, whereas the pnrB mutant degraded only 10% of the TNT in the same time period. Even after 20 days, only approximately 50% of the 0.5 mM TNT was degraded by the pnrB mutant. These results illustrate that pnrB may perform a crucial role in the TNT degradation pathway of the HK-6 strain. |
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